Chromosome breaks are the most dangerous form of DNA damage because they result in multiple types of mutations and gross chromosome rearrangements. DNA is most sensitive to breakage during replication, when hard-to-replicate noncanonical DNA structures cause replication fork stalling. Noncanonical DNA structures are strongly implicated as endogenous sources of chromosome breaks and translocations leading to developmental defects and cancers, however, the mechanisms by which replication fork stalling causes DNA double strand breaks (DSBs) are not known. Despite significant analyses of DNA damage response proteins in global or single molecule studies where the sites of damage are not identified, the molecular mechanisms of replication-dependent DNA strand breakage and repair at specific sites in human cells are incompletely understood. To address this knowledge gap, we will study two types of natural replication barriers (CTG/CAG trinucleotide repeats and asymmetric purine-pyrimidine (Pu/Py) mirror repeats) integrated at an ectopic site in the human genome where their structure and effect on replication can be manipulated. We also examine several endogenous replication fork barriers that induce DSBs during DNA replication. We will use PCR, DNA sequencing, chromatin immunoprecipitation, mass spectrometry and flow cytometry to show (1) how polymerase stalling at noncanonical DNA structures causes DSBs, (2) how DNA repair proteins act to remodel stalled replication forks to restart synthesis, and (3) the mechanisms and genomic consequences of DSB recombination at structure-induced fork barriers. We will test the hypothesis that noncanonical DNA structures induce DSB by blocking the progress of DNA polymerases, promoting nuclease-sensitive fork regression, and inhibiting DNA end processing required for recombination. Conceptual advances from this work will include determination of the molecular mechanisms of DSB formation near specific stalled forks, biochemical analysis of replication fork reversal, and identification of how the processing of structure-induced DSB differs that of nuclease-induced `clean' DSB. Our long-term goal is to define the role of DNA structure-induced g e n o m e instability in human disease. Aim 1 will disclose the relationship between fork stalling and damage signaling, the biochemistry of fork reversal, the function of structure-specific endonucleases at stalled forks, and the impact of DNA secondary structure on fork resection and repair. Aim 2 will build on our demonstration that the Fanconi anemia type J protein (FANCJ) is essential for the maintenance of noncanonical DNA structures across the genome during replication stress, to determine the mechanisms of FANCJ dependent microsatellite stabilization. In Aim 3 we will characterize the genomic consequences of FANCJ deficiency. Our experiments will show how hard-to- replicate DNA sequences cause chromosome breaks and mutations that lead to genetic disease.